Drug resistance detection method for vibrio parahaemolyticus fluoroquinolone medicines

A vibrio fluoroquinolone and detection method technology, applied in the field of pathogenic bacteria detection, can solve the problems of inability to detect hidden drug resistance of vibrio parahaemolyticus, cumbersome separation and purification of vibrio parahaemolyticus, unfavorable drug selection and treatment, etc., and achieve reduction Less preparation time, fewer reagents, and less time-consuming effect

Inactive Publication Date: 2020-05-29
HUBEI UNIV OF ARTS & SCI
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Problems solved by technology

The traditional method of drug resistance detection of Vibrio parahaemolyticus in my country is to determine the drug resistance of Vibrio parahaemolyticus by measuring the minimum inhibitory concentration or the size of the inhibition zone of Vibrio parahaemolyticus. Propagation, amplification and other steps, the detection cycle is long, the fastest will be about 48h
It is not conducive to clinical and timely drug selection and treatment
At the same time, the drug susceptibility test is to test the phenotypic drug resistance of Vibrio parahaemolyticus tentatively in vitro, and cannot detect the cryptic drug resistance of Vibrio parahaemolyticus

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  • Drug resistance detection method for vibrio parahaemolyticus fluoroquinolone medicines

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Embodiment 1

[0019] (1) Cultivate and proliferate Vibrio parahaemolyticus in shake flasks of LB medium. The culture conditions of the shake flasks are 30°C, 18h, 120rpm. After the shake flask culture is completed, 1.0g of live Vibrio parahaemolyticus cells are collected by centrifugation, and weighed with 8mL deionized water. Suspended, repeatedly freeze-thawed 5 times to break the cells, centrifuged at 12000pm for 10min, collected the supernatant, and obtained the template;

[0020] (2) Combine the 5uL template with fluoroquinolone drug resistance gene detection primers GyrA-F, GyrA-R, GyrB-F, GyrB-R, ParE-F and ParE-R (primer concentrations are 25mmol / L, respectively, with 1mM Tris-HCl--0.1mM EDTA buffer dilution) mixed together, then PCR amplification, the PCR amplification reaction system includes ultrapure water, PCR buffer, dNTP at a final concentration of 0.4mmol / L, GyrA-F with a final concentration of 0.4mmol / L, GyrA-R with a final concentration of 0.4mmol / L, GyrB-F with a final co...

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Abstract

The invention relates to a drug resistance detection method for vibrio parahaemolyticus fluoroquinolone medicines. The method comprises the following steps: (1) collecting 1.0-1.2g of live bacteria ofvibrio parahaemolyticus, performing resuspension by using 7-8mL of deionized water, performing repeated freezing and thawing to break cells, performing centrifugation, and collecting supernate so asto obtain a template; (2) commixing the template with fluoroquinolone medicine resistant gene detection primers GyrA-F, GyrA-R, GyrB-F, GyrB-R, ParE-F and ParE-R, and performing PCR (polymerase chainreaction) amplification; and (3) after PCR amplification is completed in the step (2), performing agarose gel electrophoresis and sequencing. The detection method provided by the invention is sensitive, specific and rapid, a very trace amount of target genes can be detected, and tedious vibrio parahaemolyticus culture is not needed.

Description

technical field [0001] The invention relates to the technical field of pathogenic bacteria detection, in particular to a method for detecting drug-resistant genes of Vibrio parahaemolyticus fluoroquinolones. [0002] technical background [0003] Fluoroquinolones have good antibacterial activity against G-bacteria, including Pseudomonas aeruginosa, and have certain antibacterial activity against G+ cocci. Domestic and foreign studies have shown that due to long-term and extensive use of fluoroquinolones, Vibrio parahaemolyticus has serious drug resistance to fluoroquinolones, which seriously affects the clinical efficacy. The fluoroquinolone resistance genes that have been discovered include: GyrA, GyrB, ParC, ParE and so on. The traditional method of drug resistance detection of Vibrio parahaemolyticus in my country is to determine the drug resistance of Vibrio parahaemolyticus by measuring the minimum inhibitory concentration or the size of the inhibition zone of Vibrio pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/18C12Q1/04C12R1/63
CPCC12Q1/04C12Q1/18
Inventor 黄升谋
Owner HUBEI UNIV OF ARTS & SCI
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